Project Summary/Abstract: Cell-cell communication lies at the heart of evolution of multi-cellular life: cells send specific signals to instruct their neighbors to adopt fates distinct of their own. An important class of developmental signals is encoded by the Wnt gene family. Wnt proteins interact with their cognate receptors of the Frizzled (Fzd) family to control countless developmental processes, from establishing the polarity of a single cell within a tissue to specifying the anterior-posterior body axis of an organism. Deregulation of Wnt signaling can have catastrophic consequences, including embryonic lethality, birth defects, and disease. With their diverse and potent activities in development and stem cells, Wnt proteins hold great promise as potent tools in cell and tissue engineering. The long-term objective of our research is to gain a better understanding of how Wnt proteins and their downstream signaling events influence cell fate decisions, and thereby advance technologies and treatments that specifically target Wnt signaling. Over the past years, we have made important contributions to the development of Wnts as research-grade reagents to manipulate human pluripotent stem (hPS) cells and generate cell populations with potential therapeutic value. However, significant challenges need to be addressed before the full potential of Wnts as therapeutics can be realized. In particular, despite their potent activities, many Wnt proteins remain difficult to isolate in a biologically active and stable form. Furthermore, with 19 Wnts and over 20 Wnt receptors (including Fzd1-10, Lrp5/6, Ror1/2, etc.) encoded in the mammalian genome, it is unclear how signaling specificity is established. Finally, many current attempts to target Wnt signaling in clinical settings are highly non-specific and produce complications and adverse side effects. The goal for the next five years is to leverage an innovative technology developed in our laboratory that utilizes engineered Wnt agonists, called Wnt mimetics, which exhibit superior biochemical properties compared to native Wnt proteins. These Wnt mimetics will be tested for their effects in several settings, including hPS cell self-renewal and differentiation, organoid cultures and in whole animals (mice). Furthermore, the proposed research will explore a new paradigm for how Wnt signaling specificity is established in vivo, and thereby enable the development of tools and approaches that pinpoint individual Wnt signaling pathways. These experiments will allow us to test our hypothesis that selective engagement and activation of individual Wnt receptors and receptor complexes trigger distinct signaling outputs and biological effects. The proposed research will significantly advance the field of stem cell research and tissue engineering by establishing new tools and methods to manipulate Wnt signaling in vitro and in vivo. With its abundant roles in human disorders and diseases, a better understanding of Wnt signaling is essential for the development of novel therapies for currently incurable diseases.